PROJECT SUMMARY There is now increasing evidence that pathological pain states are dependent on changes in the brain itself. Descending modulatory pathways are known to mediate top- down regulation of nociceptive processing, transmitting cortical and limbic influences to the dorsal horn of the spinal cord. Ascending pain transmission pathways are also intimately intertwined with these modulatory systems, forming positive and negative feedback loops. The output node of the best-characterized pain-modulating system is the rostral ventromedial medulla (RVM). Building on the Heinricher laboratory's experience defining the outputs of RVM neurons, the studies in the present application fill an important gap, identifying a pathway through which noxious input reaches the RVM. The RVM has two pain-modulating cell types: ?ON-cells,? which exert a net facilitating influence on nociception, and ?OFF-cells,? which have a net inhibitory action. The overarching goals of the present proposal are to understand plasticity of this circuitry in chronic pain states, and how it is modulated by endogenous opioids and cannabinoids. We recently showed that the parabrachial complex (PB) is a critical relay of acute noxious information to the RVM. We propose to test the role of the PB in regulating the activity of RVM pain-modulating neurons, elucidate how opioids and cannabinoids modulate the activity of PB-RVM synapses, and determine how this connection is altered in chronic pain states. These studies will use in vivo single-cell recording from identified RVM ON- and OFF-cells, optogenetics, and pharmacological manipulations to test the hypothesis that the PB projection to the RVM is modulated following persistent inflammation (Aim 1). Complementing this in vivo work, parallel studies under Aims 2 and 3 will use in vitro electrophysiology in an adult RVM slice with optogenetic manipulation of identified PB-RVM terminals to define the membrane mechanisms of PB-RVM synapses and understand how these synapses are modulated by cannabinoids and opioids. We will also determine how this connection is altered in chronic inflammation. By defining pathways through which noxious information reaches pain- modulating neurons at the membrane, individual neuron, and circuit level, we can begin to define how pain-modulating circuits are recruited in acute and chronic pain. This information is critical if we are ever to develop treatments addressing chronic pain as maladaptive brain disease.